Protein hydrogen exchange mechanism: Local fluctuations

Abstract

Experiments were done to study the dynamic structural motions that determine protein hydrogen exchange (HX) behavior. The replacement of a solvent-exposed lysine residue with glycine (Lys8Gly) in a helix of recombinant cytochrome c does not perturb the native structure, but it entropically potentiates main-chain flexibility and thus can promote local distortional motions and large-scale unfolding. The mutation accelerates amide hydrogen exchange of the mutated residue by about 50-fold, neighboring residues in the same helix by less, and residues elsewhere in the protein not at all, except for Leu98, which registers the change in global stability. The pattern of HX changes shows that the coupled structural distortions that dominate exchange can be several residues in extent, but they expose to exchange only one amide NH at a time. This 'local fluctuation' mode of hydrogen exchange may be generally recognized by disparate near-neighbor rates and a low dependence on destabilants (denaturant, temperature, pressure). In contrast, concerted unfolding reactions expose multiple neighboring amide NHs with very similar computed protection factors, and they show marked destabilant sensitivity. In both modes, ionic hydrogen exchange catalysts attack from the bulk solvent without diffusing through the protein matrix.Keywords: Cytochrome c; hydrogen exchange; local fluctuation; glycine mutations One wants to understand the structural and dynamic determinants of protein hydrogen exchange (HX). This problem has become important because HX methods are now able to provide direct information on protein structure, structure change, interactions, dynamics, and folding, resolved to the level of individual amino acids (Woodward 1994;Scholtz and Robertson 1995;Wand and Englander 1996).Exchange rates for hydrogens that are freely exposed to solvent in unstructured polypeptides are quantitatively predictable (Molday et al. 1972;Bai et al. 1993;Connelly et al. 1993). Rates are much slower for hydrogens that are protected by H-bonded structure, whether they are buried or at the solvent-exposed surface. The exchange of amide hydrogens is catalyzed, above pH 4, by direct attack and Hbonding of solvent hydroxide ion to the exchangeable hydrogen (Berger and Linderstrøm-Lang 1957;Eigen 1964;Englander and Kallenbach 1983). To allow for hydrogen bonding to hydroxide ion, a sizable separation of any protecting H-bond is necessary (Milne et al. 1998), requiring some transient distortion of local structure. Many dynamical models for the structural events that allow protected hydrogens to exchange have been considered. Extended cooperative unfolding reactions, both global and subglobal, have now been documented in particular cases. The suggestion that more local transient distortions determine the exchange of many other hydrogens (Bai et al. 1995;Milne et al. 1998) has become controversial (Hilser and Freire 1996;Wooll et al. 2000).We explored a mutational approach. A surface lysine residue in recombinant cytochrome c (Cyt c) was changed 153...

Structural and Dynamic Characteristics of a Partially Folded State of Ubiquitin Revealed by Hydrogen Exchange Mass Spectrometry

“…Indeed, unless each opening event exposes a set of amides to solvent simultaneously (cooperative unfolding), the observed exchange pattern will be uncorrelated even if labeling of each exposed amide is a very fast process. For example, a gradual shift of the isotopic cluster corresponding to the highly protected Ub species toward a lower m/z likely corresponds to amide exchange from the native state through local structural fluctuations (27), analogous to behavior observed for two-state proteins (11). At the same time, a gradual decline of the intensity of the isotopic cluster corresponding to the N-state and an increase in the abundance of the cluster corresponding to the A-state are indicative of a first-order transition between these two states.…”

“…This can be done in a fashion similar to that used for treatment of structural fluctuations in the native state (11) with one important exception. The full range of fluctuations within the native state (L) was assumed to be rather limited due to the presence of stable tertiary structure, since there is abundant evidence that such fluctuations primarily affect amides that are positioned close to the protein surface (27). If the A-state lacks a stable hydrophobic core (26), the range of structural fluctuations within the A-state should be unlimited (i.e., any one of the N-K protected amides can be exchanged from the A-state through fluctuations).…”

Protein structure change studied by hydrogen-deuterium exchange, functional labeling, and mass spectrometry

“…2), where HX rate (k ex ) depends on the structural opening equilibrium constant, K op , and pH, generally holds below pH Ϸ10. The term k int, the intrinsic second-order HX rate constant calibrated for unprotected amides (28,29), is applicable when amide hydrogens are freely exposed to solvent, e.g., in a concerted unfolding reaction, but perhaps not when exchange occurs by way of more constricted local fluctuations (30). When Eq.…”

The Folding Energy Landscape of the Dimerization Domain of Escherichia coli Trp Repressor: A Joint Experimental and Theoretical Investigation

“…An example of the change in HX behavior as a function of protein concentration is shown in Figure 5B. As the protein concentration was increased, the apparent number of amide hydrogens exchanging through the fast, k f , process decreased and the number exchanging through the slow, k s , process increased for peptide 26-35, segment 31-47, and peptide [42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61]. No reliable trends were observed for the k i1 and k i2 processes for these peptides or segments.…”

“…The B and C helices of [2-66] 2 TR-Only one rather long peptide, 22-47, could be generated that includes the short B helix (35)(36)(37)(38)(39)(40)(41)(42). However, this peptide also encompasses the last nine residues of the A helix, the linkers between the three helices, and the first three residues of the C helix.…”